Determining safe and effective dosages for chemotherapy drugs has been and remains a significant challenge in the treatment of cancer, particularly because overdose can be fatal (see Ames B N, Gold L S, Willet W C, “The causes and prevention of cancer”, Proceedings of the National Academy of Science, 90, 7915-7922, 1995; Dollinger M, Rosenbaum E H, Cable G, Everyone's Guide to Cancer Therapy. Kansas City, Mo.: Somerville House Books; 1994). Intercalating drugs, which are the most widely used chemotherapy drugs, prevent cell growth through incorporation into DNA and RNA, which causes improper replication of cancerous cells and incomplete biochemical synthesis. The medicinal success of intercalating drugs is based upon the higher rate of cancer cell multiplication, as compared to that of normal cells. Nevertheless, normal cell growth is also adversely affected, and that is especially true of those cells that involve rapid cell turn-over, such as blood cell production in bone marrow. These dangerous side-effects preclude the use of clinical trials to establish statistical bases for dosages, which are therefore usually derived from knowledge obtained from experience gained with limited sets of previously treated patients.
A secondary approach for determining safe and effective dosage is to monitor metabolism of the drug during administration, with concentrations being adjusted accordingly. Such information can be extremely beneficial and important, since the patient's genetic makeup and nutritional habits can strongly influence the pharmacokinetics of a drug (Ames B N, “Micronutrients prevent cancer and delay aging”, Toxicol. Lett. 102-103; 5-18, 1998). Unfortunately, current practices require the use of 10-20 mL of blood per analysis (Goodman M, Riley M B. “Chemotherapy: Principles of administration” in Cancer Nursing: Principles and Practice, 4th ed, Eds: Groenwald S L, Frogge M H, Goodman G, Yarbro C H, Jones and Bartlett: Boston, 1997), and the multiple samples that are required for profiling pharmacokinetics may further jeopardize the patient's health; consequently, they are rarely performed (Cone, E T, Jenkins A J. Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, Eds. Wong S H Y, Sunshine I, Chapter 18, CRC Press: New York, 1997).
Saliva analysis has long been considered an attractive potential alternative to the approaches referred to above, and recent research has shown that drug metabolism is often equally represented in saliva as it is in blood plasma, typically at microgram/mL concentrations (van Warmerdam L J, van Tellingen O, ten Bokkel Huinink W W, Rodenhuis S, Maes R A, Bijnen J H, “Monitoring carboplatin concentrations in saliva: a replacement for plasma ultrafiltrate measurements?”, Ther Drug Monit, 17:5, 465-470, 1995; Takahashi T, Fujiwara Y, Sumiyoshi H, Isobe T, Yamaoka N, Yamakido M, “Salivary drug monitoring of irinotecan and its active metabolite in cancer patients”, Cancer Chemother Pharmacol, 40, 449-52, 1997; de Jonge M J, J V Verwiej, W J Loos, B K Dallaire, A Sparreboom, “Clinical pharmacokinetics of encapsulated oral 9-amino-camptothecin in plasma and saliva”, Clin Pharmacol Ther, 65, 491-499, 1999; Joulia J M, Pinguet F, Ychou M, Duffour J, Astre C, Bressolle F (1999) Eur J Cancer 35:296). Analysis of saliva provides a highly desirable option, in that it is non-invasive, reduces the risk of HIV infection, is readily obtained and is relatively easy to analyze chemically. The composition of saliva is 99.5% water, and the concentrations of interfering physiological chemicals are typically at least 100 times lower than in blood plasma or urine (Chamberlain, J., The Analysis of Drugs in Biological Fluids, 2nd Ed., CRC Press (1995). Current techniques for saliva analysis, however, like that of blood, require the use of samples of 10 to 20 mL in order to enable chemical separation and detection of drugs and their metabolites, and such quantities are difficult to obtain or generate, as a practical matter.
One approach that is effectively used for measuring chemicals, at concentrations similar to those at which chemotherapy drugs are present in biological fluids, employs surface-enhanced Raman spectroscopy (SERS). The SERS method involves the absorption of incident laser photons within nanoscale metal structures, to generate surface plasmons, which then couple with nearby molecules (the analyte) to thereby enhance the efficiency of Raman scattering, by six orders of magnitude or more (Jeanmaire D L, Van Duyne R P, “Surface Raman Spectroelec-trochemistry”, J. Electroanal. Chem., 84, 1-20 (1977); Weaver M J, Farquharson S, Tadayyoni M A, “Surface-enhancement factors for Raman scattering at silver electrodes: Role of adsorbate-surface interactions and electrode structure”, J. Chem. Phys., 82, 4867-4874 (1985)). In addition to sensitivity, the rich molecular vibrational information provided by Raman scattering yields exceptional selectivity and allows virtually any chemical to be identified while also distinguishing multiple chemicals in mixtures (see Garrel R L, “Surface-Enhanced Raman Spectroscopy,” Analytical Chemistry, 61, 401A-411A (1989); Storey J M E, Barber T E, Shelton R D, Wachter E A, Can-on K T, Jiang Y “Applications of Surface-Enhanced Raman Scattering (SERS) to Chemical Detection”, Spectroscopy, 10(3), 20-25 (1995)).
Four methods have become common in the practice of generating surface-enhanced Raman scattering: (1) the use of activated electrodes in electrolytic cells (see for example Jeanmaire or Weaver above); (2) the use of activated silver and gold colloid reagents (Kerker, M., O. Siiman, L. A. Bumm, D. S. Wang, “Surface-enhanced Raman Scattering of citrate ion adsorbed on colloidal silver,” Applied Optics, 19, 3253-3255 (1980); Angel, S. M., E. F. Katz, D. D. Archibold, L. T. Ein, D. E. Honigs, “Near Infrared Surface-enhanced Raman Spectroscopy. Part II: Copper and gold colloids,” Applied Spectroscopy, 43, 367 (1989)); (3) the use of activated silver and gold substrates (Seki., H., “Surface-enhanced Raman Scattering of pyridine on different silver surfaces,” J. Chemical Physics, 76, 4412-4418 (1982) or Li, Y.-S., T. Vo-Dinh, D. L. Stokes, Y. Wang, “Surface-Enhanced Raman Analysis of p-Nitroaniline on Vacuum Evaporation and Chemical Deposited Silver-Coated Alumina Substrates”, Applied Spectroscopy, 46, 1354 (1992)); and (4) the use of sol-gels doped with silver or gold particles (Farquharson et al. U.S. Pat. No. 6,623,977, and corresponding International Application Publication No. WO 01/33189 A2, which are commonly owned herewith and the entire specification of which United States patent is hereby incorporated by reference thereto).
Surface-enhanced Raman scattering measurements have been reported for numerous drugs, including sulfa-drugs (Sutherland W S, Lasema, J J, Angebranndt, M J, Winefordner, J D “Surface-Enhanced Raman Analysis of Sulfa Drugs on Colloidal Silver Dispersion,” Analytical Chemistry, 62, 689-693 (1990)), abused drugs (Perez R, Ruperez A, Laserna J J, “Evaluation of silver substrates for surface-enhanced Raman detection of drugs banned in sports practices,” Analytical Chemica Acta, 376, 255-263, 1998; Carter J C, Brewer W E, Angel S M “Raman spectroscopy for the in situ identification of cocaine and selected adulterants,” Applied Spectroscopy, 54, 18761881 (2000), and chemotherapy drugs (Rivas L, Murza A, Sanchez-Cortes S, Garcia-Ramos J V, “Adsorption of acridine drugs on silver: surface-enhanced resonance Raman evidence of the existence of different adsorption sites,” Vibrational Spectroscopy, 25, 19-28, 2001); Nabiev I R, Morjani H, Manfait M, “Selective analysis of antitumor drug interaction with living cells as probed by surface-enhanced Raman spectroscopy,” European Biophysics Journal, 19, 311-316 (1991); Fabriciova G, Sanchez-Cortez S, Garcia-Ramos J V, Miskovsky P, “Joint application of micro-Raman and surface-enhanced Raman spectroscopy to the interaction study of the antitumoral anthraquinone drugs danthron and quinzarin with albumins,” J Raman Spectrosc 35 384-389 (2004)).
In most of the measurements made in connection with the foregoing, SERS spectra were obtained to demonstrate the ability to identify small quantities of illegal drugs (sulfa-drugs, banned sports drugs, and cocaine) or to elucidate drug structure and surface interactions in binding (acridine, danthron, etc.). In several studies the ability to analyze drugs in urine or plasma by SERS has been suggested, but not successfully performed (Ruperez A, Lasema J J, “Surface-enhanced Raman spectrometry of triamterene on a silver substrate by the nitric acid etching method,” Talanta, 44, 213-220 (1997); Farquharson S, Lee Y H “Trace drug analysis by surface-enhanced Raman spectroscopy,” SPIE 4200: 89-95, (2000); Eliasson C, Lore A, Murty K V G K, Josefso M, Kail M, Abrahamsson J, Abrahamsson K, “Multivariate evaluation of doxorubicin surface-enhanced Raman spectra,” Spectrochimica Acta Part A 57: 1907-1915 (2001)). In the work of Farquharson and Lee, the SERS spectrum of a urine sample from a chemotherapy patient was reported but, due to the presence of dominating obscuring peaks (e.g., of uric acid and creatinine) in the spectra, no drug was identified or quantified. In any event, no known prior art describes or suggests the use of saliva to detect drugs and/or their metabolites by surface-enhanced Raman spectroscopy, albeit papers recently published do allude to such methodologies (Gift A, Shende C, Inscore F, Maksymiuk P, and Farquharson S, “Five minute analysis of chemotherapy drugs and metabolites in saliva: Evaluating Dosage,” SPIE 5261: 135-141 (2004); Farquharson S, Shende C, Inscore F, Maksymiuk P, and Gift A, “Analysis of 5-fluorouracil in saliva using surface-enhanced Raman spectroscopy,” J Raman Spectrosc, accepted (2004); Farquharson, S, Inscore, F E, Maksymiuk, P, Gift, A, Shende, C, “Medical applications of surface-enhanced Raman spectroscopy,” Analytical Bioanalytical Chemistry, submitted, September 2004).